Project Summary Nucleic acids serve important hereditary and regulatory roles within cells, and the optical imaging of nucleic acids has led to many insights on the behavior of biological systems. Current in situ and in vivo methods for nucleic acid imaging are limited in their sensitivity, quantitative precision, specificity, and multiplexing. DNA nanotechnology can, in principle, improve bio-imaging performance in all four categories, but conventional DNA nanotechnology requires thermal annealing and cannot easily be applied to biological systems. In this proposal, DNA and RNA nanostructures and nanodevices that assemble and operate isothermally are presented and tested as bio-imaging tools. For in situ whole embryo mRNA imaging, geometrically precise DNA nanostructures will act as bright optical tags specific to each mRNA target of interest. Each DNA nanostructure tag has a precise number of functionalized fluorophores, so fluorescence can be directly mapped to concentration or copy number. Furthermore, the large number of fluorophores colocalized to each target molecule will facilitate imaging by reducing microscope sensitivity requirements. For live cell and organism imaging, two different approaches are proposed. The first approach ensures highly specific imaging using a recently developed molecular mechanism for mimicking melting temperature conditions across a range of temperatures, salinities, and concentrations. By adopting this mechanism to fluorescent nucleic acid probes microinjected into living cells, highly specific imaging of endogenous nucleic acids can be achieved. This is particular relevant for imaging microRNAs, short RNA molecules that play important regulatory roles inside the cell, that often differ from other microRNAs by as little as a single base pair. The second, potentially much more powerful, approach is the construction of an genetically encoded allosteric RNA nanodevice. When an endogenous target RNA molecule binds to the RNA nanodevice, the nanodevice reconfigures to reveal an aptamer that activates the fluorescence of a GFP-based conditional fluorophore. The conditional fluorophore is small enough to diffuse into living cells, so it will be possible to image endogenous RNA without the use of any exogeneously introduced probes. Initial in vitro studies have yielded promising results. Isothermally assembled DNA nanostructures in both native and denaturing conditions have been verified by gel electrophoresis, atomic force microscopy, and total internal reflection fluorescence microscopy, and studies will shortly being on the in situ imaging of whole Drosophila Melanogaster (fruit fly) embryos. The mechanism for ensuring high specificity nucleic acid hybridization has been demonstrated across a variety of temperatures and salinities, and a typical single-base change in target sequence causes hybridization to be impaired by a factor of 26.